Not only are many susceptible North American insectivorous bats nearing extinction and, thus, scarce for experimental studies, but they are difficult to care for and maintain in captivity because of their specialized habitats and diets. In this study, we explored porcine ears as a potential substrate for studying infection development and the dynamics of P. destructans growth in the laboratory. Porcine ear skin shares many tissue-level similarities with bat skin and is a readily available resource. We found the porcine ear model provided a substrate faithfully mimicking external P. destructans colony morphology and internal histology similar to what is seen with P. destructans infections in bat wing membranes. This model provided a major advance by distinguishing virulence attributes between a wild-type Pseudogymnoascus destructans strain harboring a partitivirus common to all North American strains of the fungus and an isogenic virus-cured P. destructans strain. ImageJ analysis showed that the cured P. destructans strain was reduced significantly in ability to produce hyphal cover and showed less spore production on porcine skin. Taken together, these results strengthen our previous finding that the partitivirus infection has a role in WNS and provides a valuable model host tool in understanding P. destructans virulence factors for therapeutic application.White-nose syndrome (WNS), responsible for the mass mortality of North American bats, lacks economically viable and practical IMPORTANCE This work describes an important insight into the role of Pseudogymnoascus destructans partitivirus in fungal biology and provides a model system for studying white-nose syndrome in bats, which has decimated North American populations. Pseudogymnoascus destructans is a filamentous, psychrophilic fungus responsible for causing white-nose syndrome (WNS) in North American bats , that has been implicated in some aspects of the fungal biology . For this study, we did not use any of the drugs that are known mutagens but applied the same PEG method with serial passaging of our wild-type P. destructans isolate, LB01, over three generations in PEG at −4 MPa in minimal nutrition media. LB01 and three of these virus-free cultures were sequenced using a next-generation sequencing platform by the laboratory of Kurt Lamour, Lightening Genomics, followed by SNP analysis. SNP analysis showed only a few variations in LB01, LB01-PEG3-1#4, and LB01-PEG3-2#4 (P. destructans isolate 20631-21 sequence in GenBank (accession no. KV441386 and LAJJ00000000) , producing matric potentials in a range from −2 MPa to −4 MPa in minimal nutrient medium . HoweverPEG3-2#4  comparedP. destructans growth on the porcine ear tissue on the sixth day postinoculation , elongation factor (EF), and glyceraldehyde-3-phosphate dehydrogenase (gpd) genes on random P. destructans colonies growing on the porcine ear surface , elongation factor (EF), and glyceraldehyde-3-phosphate dehydrogenase (gpd) from DNA extracted from the fungal colonies growing on the surface of porcine ears postinoculating spore suspension of LB01 isolate and its virus-free cultures of Copyright © 2022 Thapa et al.2022Thapa et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. destructans hyphal mats growing on the epidermal layer of the skin and bearing profuse curved conidia characteristic of P. destructans. The epidermal layers were ruptured in many places and the fungus was found growing in cup-like depressions  control plates  in ImageJ   .P. destructans infection and development. We tested the model using two isogenic virus-free P. destructans cultures to evaluate the response of P. destructans on the porcine ear. We found the porcine model to mimic infections observed in bat tissues  of hyphal mass in the virus-free compared with the virus-infected treatments. In our previous study, we showed that the virus-free isolate spread on the plates at the same rate as the virus-infected isolates , LB01-PEG3-2#4 (2#4), and LB01-PEG3-1#2 (1#2), derived from LB01 after polyethylene glycol (PEG) treatments. The two dsRNA bands in LB01 lanes correspond to dsRNA1 and dsRNA2 of PdPV-pa. Although LB01-PEG3-1#2 is virus free, it shows a higher number of single nucleotide variations; thus, it was not used in this study. Lane M is the lambda DNA marker digested with EcoRI and HindIII. Download FIG S1, PDF file, 0.4 MB.Double-stranded RNA profile of Copyright © 2022 Thapa et al.2022Thapa et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Ears were collected from freshly sacrificed animals collected from the Penn State meat laboratory. Immediately after slaughtering, the animals were scalded in boiling water for hair removal and surface sterilization. The ears were then harvested using clean knives, wearing gloves, and transferred to the laboratory in a sterile plastic bag on ice. All further manipulation was done in a class II, type B2 biological safety hood. The ears were removed from the bag using sterile surgical gloves and placed in a sterile glass container. One of the ears was cut into four pieces, two pieces from the top half and two pieces from the bottom half, using a sterile surgical scalpel blade and sterile surgical scissors . The oth10.1128/msphere.01022-21.2FIG S2FIG S2, PDF file, 0.3 MB.Porcine ear cut into pieces (A) and the intact porcine ear (B) used in the experiment. Download Copyright © 2022 Thapa et al.2022Thapa et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. destructans conidial suspension of approximately 2.5 × 108 conidia/mL was spread with the help of sterile pipette tips, and the conidia were allowed to settle on the surface by keeping the ear in a position that prevented overflow for about 2 min. The conidial load was determined based on the production of conidia over a 3-week period under similar initial load for the virus-free and wild-type isolate. In the experiment, we used the lowest possible load that resulted in colonization on culture plates for both the virus-free and wild-type isolates. The conidia of the wild-type P. destructans that had a natural infection of PdPV-pa (LB01) and the virus-free cultures of P. destructans (LB01-PEG3-1#4 and LB01-PEG3-2#4) were collected from 3-week-old Sabouraud dextrose agar (SDA) culture plates grown under conditions described previously , elongation factor (EF), and glyceraldehyde-3-phosphate dehydrogenase (gpd) regions, followed by sequence analysis to confirm P. destructans . We used reagent stations with an ethanol series , histosolve, and paraffin where the tissues were automatically treated over seven and a half hours. The total time allocated was 30 min in each ethanol series, histosolve for 40 min, and 45 min in paraffin. Some of the ethanol series, histosolve, and paraffin treatments were repeated automatically .The tissue was removed from the cassette and embedded in paraffin using a Leica EG1150C. The tissue was kept upright while embedding in paraffin so that its transverse section was taken. After properly embedding the tissue in the correct orientation in paraffin and cooling it enough to settle the paraffin over the cold plate of the Leica EG1150C, the tissue was sectioned using a Thermo Scientific microtome (Shandon Finesse 325 microtome) into 10-μm sections and floated in a luke-warm water bath. Once any wrinkles in the sections disappeared, they were removed by positioning a slide under each section and gradually lifting it out of the water. The slides were then dried over a slide warmer for about 30 min.The tissue section was stained with hematoxylin and eosin in an automatic stainer (Leica Autostainer XL). The process took 55 min. The tissue section was then mounted with xylene substitute on the slide. Once the slides dried, the sections were observed under a microscope (Olympus BX60), and photographs were taken.The tissue fixation, sectioning, and staining were done at the core Microscopy Facility of Huck Institutes of Life Sciences, Pennsylvania State University, University Park, PA.P. destructans and the isogenic virus-free strains were analyzed by applying ImageJ (t test at a 95% confidence level.The images of porcine ear tissue sections colonized with wild-type g ImageJ . We usedAll data reported in this paper are available on request.


Since hearing loss and cognitive decline often co-occur among older adults, a cognitive screening test suitable for hearing-impaired people is of high clinical relevance. We report the first evaluation of a German language version of the Montreal Cognitive Assessment—Hearing Impaired version (MoCA-HI).The aim of the present study was to compare cognitively healthy participants with and without hearing loss, to examine the impact of age, sex, educational level and degree of hearing impairment on the German MoCA-HI performance, and to develop normative data.The German MoCA-HI was tested in 94 participants with normal or mild hearing impairment (group 1: 4PTA ≤ 40 dB on the better hearing ear) and 81 participants with moderate to profound hearing loss (group 2: 4PTA > 40 dB on the better hearing ear). Additionally, all participants performed the standard MoCA (version 8.2).p = 0.05). In contrast, group 1 performed significantly better than group 2 on the standard MoCA (p < 0.001). There was no difference between the MoCA and the MoCA-HI performance in group 1 (p = 0.12), whereas individuals of group 2 performed significantly better on the MoCA-HI than on the standard MoCA (p < 0.001). Test-retest reliability of the MoCA-HI was high (p < 0.001). Higher age (p < 0.001), male sex (p = 0.009) and lower education (p < 0.001) were associated with a lower overall MoCA-HI score. Based on the demographic data normative data were developed by a regression-based approach.No significant group difference between group 1 and 2 was found in the MoCA-HI total score (The MoCA-HI is a cognitive screening test which is suitable for people with hearing impairment. Age-related hearing loss and dementia are among the most common chronic diseases in old age. Currently, approximately 430 million people live with disabling hearing loss , while 5There is a growing interest in neurocognitive testing in settings outside psychologic or psychiatric ones , 10, parNumerous screening tests are available to identify cases of cognitive impairment . HoweverSeveral attempts have been made to adapt cognitive screening tests for people with hearing loss , 22. Adawww.mocatest.org). This MoCA-HI is freely available from the MoCA website (www.mocatest.org) to appropriately trained persons after a short fee-based online training offered by the same website. In Dawes et al.'s version, the spoken items of the standard MoCA (version 8.1) were presented visually  or substituted with alternative visual tasks . These adaptations were designed to index the same cognitive domain as the standard items and to be of a similar level of difficulty.Dawes et al. developed a visual version of the Montreal Cognitive Assessment (MoCA)  for peopDawes et al.'s validation was carried out using an English version of the MoCA-HI . PoolingThe aim of the present study was (1) to compare performance of the German version of the MoCA-HI and the original MoCA in cognitively healthy participants with and without hearing loss, (2) to examine the impact of age, education, sex and level of hearing loss on performance and (3) to derive corresponding performance norms of the German MoCA-HI.Inclusion criteria were as follows: (1) age of 60 years or older, (2) native or excellent German speaker, (3) normal or corrected near visual acuity of ≤ 0.3 logMAR, (4) normal performance in the GPCOG   a score. Particit-test at an alpha level of 0.05 with a power (1-beta) of 0.90, two groups of at least 70 participants were required. In total, 175 participants were included . Descriptive statistics including mean (M) and standard deviation (SD) were used to describe sociodemographic, audiological and cognitive data. T-tests were performed to compare group 1 to group 2 with regard to age, education, MoCA-HI total-score and the individual cognitive subdomains and reported by mean difference (MD) and p-value. To compare the results of the two groups in the adapted tasks of the MoCA-HI and the corresponding tasks of the standard MoCA, the Mann-Whitney-U-test was used. To examine performance differences between the MoCA and the MoCA-HI within each group, the Wilcoxon signed rank test was applied. In order to analyze the impact of hearing impairment on the MoCA-HI-total-score and the cognitive subdomains, multiple regression analysis was carried out including the 4PTA as a continuous measure of hearing ability taking into account age, education and sex. Test-retest-reliability was determined by a Pearson-correlation of the MoCA-HI total-scores at both measurement time points.To achieve a medium effect size for a group comparison using a th response from a model that was estimated without this observation  was developed using the z-score formula z = (score–expected score)/residual standard deviation. Cutoff scores were developed based on the z-score-formula for the 10th percentile (z = −1.28) for men and women for each age (60–97 years) and all years of education (7–18). Statistics were calculated by the statistical program SPSS (Version 28) and normative data were calculated by Rstudio (2021.09.1). Confidence interval was set at 95% and statistical significance was defined as a www.mocatest.org). The study met the guidelines of the Declaration of Helsinki, and all participants gave their written consent. All examiners underwent training as required by www.mocatest.org.The study was registered on the MoCA homepage , 60 subjects between 72 and 83 years  and 15 subjects were aged 84 years or older . According to the WHO definition, 52 patients did not suffer from hearing loss  dB), 42 participants were classified as WHO 1 with a mean 4PTA of 30.63 (SD 3.43) dB, 41 were suffering from a hearing loss of 45.70 (SD 5.66) dB in mean (WHO 2). 40 subjects with a mean 4PTA of 88.75 (SD 22.05) belonged to WHO 3 and 4.One hundred seventy-five participants aged 60 to 97 years  were included in the present study. 100 subjects were aged between 60 and 71 years ] and had a lower educational level than group 1 (p < 0.001) (Study samples were divided into 2 groups. Group 1 (94 subjects) was normal hearing or only slightly hearing-impaired  with a mean 4PTA of 22.23 dB (SD 8.78) and group 2 (81 subjects) was moderate or profound hearing-impaired  with a mean 4PTA of 66.96 dB (SD 26.87). Audiometric data are shown in < 0.001) .p = 0.05). However, Welch test showed that group 1 performed significantly better than group 2 in the cognitive subdomain of visuospatial and executive functions . None of the other cognitive subdomains, such as naming , attention , language , abstraction , and recall , showed a significant difference between the two groups in the independent sample t-test. For the cognitive subdomain of orientation, Welch's test also showed no significant group difference .Mean scores of the MoCA-HI and the subdomains in group 1 and group 2 are shown in p < 0.001), male sex , and lower education  were associated with a lower MoCA-HI total score and together explained 41.7% (adjusted R2 = 0.41) of the total variance . Mean total scores in the MoCA-HI in the 4 different WHO groups are shown in p = 0.72). Further no significant effect of the level of hearing impairment on the different subscores was found .A multiple linear regression analysis revealed that older age . However, the MoCA-HI total score was higher in the retest than at baseline , due to a statistically significant improvement in the cognitive subdomain “recall” in the retest (p < 0.001). All other subtests remained stable after re-testing.A retest was performed in 115 participants. The mean retest interval was 60.38 (SD 18.08) days after the first administration with a minimum of 28 and a maximum of 112 days. Test-retest reliability was high with a Pearson correlation of 0.84 (p = 0.58). In contrast, individuals of group 1 (mean rank = 106.27) performed significantly better than subjects of group 2 (mean rank = 66.80) on the sum of the corresponding items of the standard MoCA (p < 0.001). Further, group 1 did not differ in the MoCA and MoCA-HI performance (p = 0.12), whereas group 2 performed significantly better on the MoCA-HI than on the standard MoCA (p < 0.001).There was no difference between group 1 (mean rank = 89.70) and group 2 (mean rank = 86.03) in the adapted items of the MoCA-HI (www.mocatest.org), showing that 35.5% of women and 39.4% of men scored below the original cutoff . This effect is strongest for education (t = 7.52), followed by sex (t = −2.65), age (t = −1.86), and the interaction of age and sex (t = −1.41), as indicated by the t-values. Based on the present data, the z-Score could be determined as follows: z = (Score–(22.86 + (−0.07 * age) + (0.53 * education) + (−1.11 * sex) + (−0.07 * (age−71.52) * sex)))/2.72. Sex was coded as 0 = female and 1 = male, age and education are inserted in years. The resulting cutoff scores for the 10th percentile are shown in In a first step, MoCA-HI scores were adjusted for education as suggested by Dawes  from the assessment and established new cutoff scores proportionally adjusted for the deleted items . Howevern = 103) vs. hearing loss (n = 49)  . Lerch a(n = 49) . A compa(n = 49) . Utoompr(n = 49) , 30 cognThe visual version of the MoCA reported in the current study has several advantages over previous versions of the MoCA adapted for people with hearing loss. First, rather than deleting spoken items, it replaces the standard spoken items by other items that tap into the same cognitive domain and are of a similar level of difficulty. Secondly, it was validated in a large cohort in a multi-centre study , 26. ThiGroup 2 (hearing loss of ≥ 41 dB) performed worse than group 1, which had no hearing loss or only a mild hearing loss, on the standard MoCA, but not on the three adapted tasks of the MoCA-HI. In line with that, there was no significant difference in the performance in the MoCA and MoCA-HI of group 1, while group 2 performed significantly worse in the standard MoCA than in the MoCA-HI. Thus, at least people with a severe hearing impairment may benefit from a visual version of the MoCA and the MoCA-HI may prevent false-positive diagnosis of dementia especially in case of a severe or profound hearing loss . HoweverThe MoCA-HI total score was best predicted by a regression model including age, education, sex, and the interaction of age and sex; age had a stronger effect on the total score in men than in women. This is in line with previous studies on the original MoCA, where regression models including age, education and sex had the best predictive power , 40, 41.To use the MoCA-HI in clinical practice, a re-test is necessary. In the present study participants performed slightly better in the re-test with less than 1 point more. Although this improvement was statistically significant, it did not make a difference to the clinical classification on the MoCA-HI. Practice effects cannot fully be ruled out in re-testing , althougOne limitation of our study is that we relied on the GPCOG to establish normal cognition criteria. The GPCOG is somewhat like the standard MoCA in including spoken items, so hearing status may have impacted categorization as normal cognition based on GPCOG performance. Some people with hearing loss might have been incorrectly excluded. However, we do not consider this to be a serious issue, since it was our aim to include only cognitive healthy individuals in this analysis.Previous research indicated that performance criteria to identify cognitive impairment developed in English may not be applicable to translations of the MoCA in other languages . Cut-poiThe German translation of the MoCA-HI is suitable in subjects with and without hearing loss and has high retest reliability. Performance criterion for identification of cognitive impairment should be developed, considering the impact of age, sex and educational level. A language-specific validation is required due to linguistic and cultural differences.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The present study was approved by the Ethic Institution of the Ruhr-University Bochum (No. 20-7076). The patients/participants provided their written informed consent to participate in this study.CV, LG, and PD designed the study. HF collected the data and did the statistical analysis with critical feedback from FL and MT. CV and HF wrote the manuscript with contributions from PD and critical feedback from LG, ZN, and RW. All authors contributed to the article and approved the submitted version.


The good anti-corrosion performance was fascinatingly observed in salt spray tests even without obvious corrosion phenomenon after 30 days of testing. Due to these remarkable properties, GPP nanocomposites can be an outstanding candidate for the rapid development of broadband shielding and anticorrosive materials.To address the challenging issues of metal materials corrosion in industries, which has caused huge economic losses and security threats to many facilities in marine environments, functional polymer coatings have been widely used and regarded as one of the simplest and most effective methods to prevent such an undesirable event. In this study, a new type of coating filler consisting of graphene oxide/polyaniline/polydopamine (GO-PANI-PDA) nanocomposites has been successfully synthesized. The morphology, structure, composition, and corrosion resistance performance of the GO-PANI-PDA (GPP) nanocomposites were investigated via a series of characterization methods. The results from our electrochemical impedance spectroscopy, potentiodynamic polarization curve and salt spray experiment showed that the best corrosion resistance performance of the coating is from GPP 21 with the epoxy/GO-PANI:PDA ratio of 2:1, which exhibited a positive corrosion potential (−0.51 V) shift from epoxy/GO-PANI coating (−0.64 V). The corrosion current density (3.83 × 10 Over past few decades, many effective strategies such as adding corrosion inhibitors, cathodic protection, surface treatment and coating technology have been widely developed to prevent corrosion in the marine industry and ensure the structural integrity of the alloy ,2,3. Amo2O, O2 and Cl− will inevitably penetrate into its coating layers and reach the substrate surface through holes and defects formed by solvent volatilization during long-term immersion [2, SiO2 and graphene into epoxy coatings to improve their anti-corrosion properties [Epoxy resin (EP) has good mechanical properties, considerable adhesion and stable chemical properties. However, corrosive media such as Hmmersion . One of operties ,10,11,12operties ,16,17,18operties ,20,21. Moperties ,23. DeBeoperties  found thoperties  and Zhanoperties  added PAoperties . Althoug− in GO and -NH3+ in PDA, we propose that the grafting of dopamine on GO will help promote the compatibility of GO-PANI nanosheet in epoxy resin, thereby improving the corrosion inhibition efficiency of the coating. Nastaran et al. [The amino and hydroxyl functional groups of dopamine (DA) make it easy to be functionalized and cross-linked with other materials, resulting in the improvement of the dispersion ability and the compatibility between fillers and resins ,31,32,33n et al.  preparedn et al.  confirmeIn this paper, GO-PANI nanosheets were functionalized with PDA to fabricate GO-PANI-PDA(GPP) nanocomposites by in situ chemical polymerization of dopamine (DA) in alkaline environment. The structure and morphology of GPP nanocomposites were characterized by scanning electron microscope (SEM), Fourier-transform infrared spectroscopy (FT-IR), and X-ray diffraction (XRD) measurements. The anti-corrosion performance of EP/GPP coatings were tested by electrochemical methods and salt spray experiments. The mechanical properties, hydrophilic and hydrophobic properties, and adhesion properties of the coatings were tested by impact resistance, contact angle and adhesion tests. The GO-PANI-PDA (GPP) coating we prepared here will not only play the sheet barrier role of GO prolongs the diffusion path of corrosive substances, but also avoid forming galvanic corrosion.2H5OH, 99.5%) was obtained from Linshi Chemical Reagent Co., Ltd. . Aniline (AN) and ammonium persulfate (APS) were purchased from Sinopharm Chemical Reagent Co., Ltd. . Epoxy resin (E−44) and curing agent (polyamide 650) were supplied from Liangli Electronic Commerce Co., Ltd. . The deionized water was used throughout the experiments.GO was purchased from Shenzhen Tulingjinhua Tech. Co., Ltd. . Dopamine was procured from Bomei Biotechnology Co., Ltd. . Xylene was obtained from Yongbang Chemical Technology Co., Ltd. . Acetone was supplied from Jiujia Chemical Co., Ltd. . Concentrated hydrochloric acid (HCl) was obtained from Shenzhen Qihongyuan Technology Co., Ltd. . Sodium chloride was procured from Yatai United Chemical Co., Ltd. , Ethanol (CThe synthetic route of GO-PANI-PDA(GPP) nanocomposites was shown in Then, 250 mL deionized water was added in a 250 mL beaker, followed by adding 1500 mg of GO-PANI and PDA in solution and stirring for 12 h (pH 8.5 in tris buffer solution). After standing for 24 h, the GO-PANI-PDA (GPP) nanocomposites were obtained by filtering and washing with deionized water and anhydrous ethanol several times, and dried under vacuum, at 60 °C, for 24 h. The products with varying weight ratio of 4:1, 2:1, 1:1, 1:2 and 1:4 between GO-PANI and PDA were prepared and named as GPP 41, GPP 21, GPP 11, GPP 12 and GPP 14, respectively. The control sample PDA without incorporating GO-PANI was also prepared for comparison.2) and plate with a wire bar coater (200 μm and 100 μm) and cured for 24 h and then dried, at 60 °C, for 24 h to give corresponding coatings. It should be noted that the working electrode was polished with 500-mesh, 1000-mesh and 2000-mesh sandpapers in sequence and washed with alcohol to obtain clean and smooth surface before coatings. The coatings by GO-PANI, PDA, GPP41, GPP21, GPP11, GPP12 and GPP14 were prepared similarly and noted as EP/GP, EP/PDA, EP/GPP41, EP/GPP21, EP/GPP11, EP/GPP12 and EP/GPP14, respectively.The procedure for coating preparation was shown in −1. The crystal structures of prepared materials were characterized by X-ray diffraction (XRD) with a PANalytical X′Pert Pro X-ray diffractometer. The electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization measurements were adopted to detect the corrosion protection performance of each coatings in 3.5% NaCl solution, at room temperature. EIS tests were carried out at an CS165 electrochemical workstation  with a three-electrode system including the working electrode (steel electrode), the counter electrode (platinum electrode) and reference electrode (Ag/AgCl Electrode). The frequency was in the range of 105–10−2 Hz with an amplitude of ± 5 mV. The potentiodynamic polarization curves were obtained with the sweep rate of 0.5 mV/s. The corrosion resistance test of the coatings was carried out for continuous 30 days with the salt-spray test chamber  [The surface morphology of the prepared materials was characterized by scanning electron microscope (SEM)  and the surface functional groups were analyzed by Fourier-transform infrared (FT-IR) spectroscopy with a scanning range of 4000~500 cm, China) . The adhTo analyze the microstructure of the prepared nanocomposites, SEM images were taken. Some pores and agglomeration defects were present in the epoxy coatings filled with GO-PANI (GP) in θ = 15.2°, 20.3°, 25.2°, and 26.5°, corresponding to (011), (020), (200) and (121) planes, respectively. The XRD pattern of GO-PANI-PDA nanocomposites is similar to that of GO-PANI, which proves that the modification of PDA does not change the crystal structure of GO-PANI [As presented in  GO-PANI ,43,44. A−1 and 1121 cm−1 are related to the stretching vibration of N-H bond and C-N bond, respectively. The peak at 3411 cm−1 is caused by the O-H stretching vibration of GO-PANI-PDA [−1, which shifts to 1587 cm−1 for GO-PANI-PDA nanocomposite. Moreover, the characteristic peaks of PDA observed at 1343 cm−1 and 1587 cm−1 also appear in the FT-IR spectrum of GO-PANI-PDA. All these results indicates that PDA has been successfully attached to GO-PANI.The FT-IR spectra of PDA, GO, GO-PANI and GO-PANI-PDA are shown in PANI-PDA . The C=CThe electrochemical impedance spectroscopy (EIS) and potentiodynamic polarization curves were applied to investigate the corrosion resistance of EP/GP, EP/PDA, EP/GPP41, EP/GPP21, EP/GPP11, EP/GPP12 and EP/GPP14 coatings. The reference electrode was an Ag/AgCl electrode, which was calibrated before use to ensure that the same reference potential for each experiment. The working electrode was placed in 3.5 wt% NaCl solution for 3600 s to test the stability of the open circuit potential . When the is the solution resistance/electrolyte resistance, Rpo is the coating resistance, Rct is the charge transfer resistance, Cζ is the coating capacitance, Cdl is the electric double-layer capacitance/film interface constant phase element [ct of coating corresponded to good corrosion resistance. The value of Cζ can reflect the permeability resistance of the coating and the value of Cdl corresponds to the defects in coatings, and the wet adhesion between the coating layer and the metal. For the EP/GPP 21 coating, the values of Rct and Rpo reached 5.279 × 1016 Ω·cm2 and 3.183 × 106 Ω·cm2, respectively, indicating the coating has good corrosion resistance, whereas the values of Cζ and Cdl are as small as 7.257 × 10−10 Ω·cm2 and 6.071 × 10−9 Ω·cm2, respectively, indicating that a dense layer of PDA pre-formed on the surface of the substrate. It can be reasonably concluded that GPP nanocomposites can fill the defects of epoxy resin in addition to the physical barrier effect of the coating with the adhesion properties of PDA. element , msd rep−8 A/cm2) is also nearly an order of magnitude lower than that of EP/GP (7.05 × 10−7 A/cm2), and the coating exhibits the smallest current density during cathodic polarization, corresponding to the corrosion rate of 4.50 × 10−4 mm/year is also an order of magnitude lower than that of EP/GP of 8.27 × 10−3 mm/year. These results indicated that the passivation zone was formed and EP/GPP21 coating has the best corrosion resistance. The similar results from Tafel analysis also suggested the same conclusion as that obtained by the EIS analysis.The polarization experiments of EP/GPP nanocomposite coatings in 3.5 wt% NaCl solution were carried out. The corresponding Tafel curves and specific data are shown in The low values of Icorr and Ecorr exhibited by EP/GPP can be attributed to the surface-adsorbed GO-PANI-PDA molecules, and the addition of uniformly dispersed GO-PANI-PDA nanosheets can compensate for the defects in the epoxy resin matrix and improve the dispersibility and interfacial adhesion of GO-PANI nanoparticles to epoxy resin. It also further prevents the steel from being corroded by corrosive medium through the “labyrinth effect” of corrosive ion penetration, which significantly reduces the CR of GO-PANI-PDA and improves the corrosion resistance of the coating. As a result, the EP/GPP21 has a highest corrosion inhibition efficiency. It is worth noting that the thickness of the electrode coating is very critical when making the electrode coating, and finding a more accurate coating thickness control method will be of great help to the experiment.For the salt spray tests, each sample was coated with two thicknesses of 100 μm and 200 μm, and placed in a salt spray test box after edge sealing. Further salt spray experiments were performed on the EP/GPP21 coatings with different thickness (100 μm and 200 μm). The results over the salt spray test duration are as shown in Next, to evaluate the adhesion performance of the coating, we applied sufficient force to completely break the coating and check the scratch on the surface of the substrate to see whether the nearby coating falls off. −1 and 803 Ω·cm−1, respectively. After polymerization, the resistivity of GO-PAN-PDA is close to that of PDA, which is much larger than that of GO-PANI. This result suggests that the addition of PDA to GO-PANI not only maintains the sheet-like barrier properties of GO-PANI, but also increases the resistivity of the material and reduces the corrosion current, thereby enhancing the anti-corrosion performance of the composite. However, when the amount of doped PDA is small, the resistivity is relatively large. It can be reasonably explained that the excessive addition of PDA in the anti-corrosion performance test decline the anti-corrosion performance. It is worth noting that although the less PDA is added, the higher the resistivity is, a certain amount of PDA is also required to support the compatibility and adhesion between materials.It can be seen from The hydrophilicity and hydrophobicity of the coating surface were determined by contact angle in the presence of water. As shown in −8 A/cm2) was also nearly an order of magnitude lower than that of EP/GP (7.05 × 10−7 A/cm2), which corresponded to the lowest corrosion rate of 4.50 × 10−4 mm/year. The salt spray test displayed that EP/GPP 21 coating had over 30-day long-term preservative effect on Q215 steel. Doping GO-PANI in combination with the excellent properties of PDA gives GO-PANI nanosheets better compatibility and dispersion in epoxy resins, strengthening the bonding performance between the coating and the substrate. It can also adjust the electrical conductivity of the material, fill the defects in EP/GP and improve the barrier properties and electrochemical passivation properties of GO-PANI. This work is expected to be widely used in the field of long-term corrosion protection, providing the necessary protection for various marine installations, ships and other metal components.GO-PANI-PDA nanocomposites were successfully synthesized as an ideal filler to improve the anticorrosion properties of epoxy coatings. The GO-PANI-PDA nanocomposites were characterized by FT-IR, SEM and XRD, which proved that the PDA and GO-PANI were completely incorporated without changing the structure of GO-AN nanocomposites. The corrosion resistance behavior and mechanism of the EP coatings filled with GO-PANI-PDA nanocomposites were systematically studied by EIS, Tafel polarization curves and salt spray experiments. The results showed the EP/GPP21 coating had the best corrosion resistance with the positive corrosion potential (−0.51 V) shifted compared with that of the EP/GP coating (−0.64 V). The corrosion current density (3.83 × 10


Deep learning-based denoising is promising for myocardial perfusion (MP) SPECT. However, conventional convolutional neural network (CNN)-based methods use fixed-sized convolutional kernels to convolute one region within the receptive field at a time, which would be ineffective for learning the feature dependencies across large regions. The attention mechanism (Att) is able to learn the relationships between the local receptive field and other voxels in the image. In this study, we propose a 3D attention-guided generative adversarial network (AttGAN) for denoising fast MP-SPECT images.99mTc-sestamibi stress SPECT/CT scan were retrospectively recruited. Sixty projections were acquired over 180° and the acquisition time was 10 s/view for the full time (FT) mode. Fast MP-SPECT projection images (1 s to 7 s) were generated from the FT list mode data. We further incorporated binary patient defect information  into AttGAN (AttGAN-def). AttGAN, AttGAN-def, cGAN, and Unet were implemented using Tensorflow with the Adam optimizer running up to 400 epochs. FT and fast MP-SPECT projection pairs of 35 patients were used for training the networks for each acquisition time, while 5 and 10 patients were applied for validation and testing. Five-fold cross-validation was performed and data for all 50 patients were tested. Voxel-based error indices, joint histogram, linear regression, and perfusion defect size (PDS) were analyzed.Fifty patients who underwent 1184 MBq All quantitative indices of AttGAN-based networks are superior to cGAN and Unet on all acquisition time images. AttGAN-def further improves AttGAN performance. The mean absolute error of PDS by AttcGAN-def was 1.60 on acquisition time of 1 s/prj, as compared to 2.36, 2.76, and 3.02 by AttGAN, cGAN, and Unet.Denoising based on AttGAN is superior to conventional CNN-based networks for MP-SPECT. Myocardial perfusion single photon emission computed tomography (MP-SPECT) is a standard method for the quantitative diagnosis of coronary artery disease (CAD) . HoweverImage noise is a substantial problem for fast MP-SPECT due to the limited detected counts and the fact that it degrades the image quality, hampering clinical diagnosis and quantification results . RecentlHowever, conventional CNN-based methods use fixed-sized convolutional kernels to convolute one local region within the receptive field at a time, which would be ineffective for learning the feature dependencies across large regions . The fea99mTc-sestamibi injection on a clinical SPECT/CT system  were retrospectively enrolled in this study under local ethics approval  algorithm with 5 iterations and 4 subsets, with CT-based attenuation and dual energy window scatter corrections. The reconstruction matrix size was 64 × 64 × 19 with a voxel size of 0.6096 cm. A 3D post-reconstruction Gaussian filter with a standard deviation of 0.6 voxel was applied on the FT images for data analysis.The architecture of the AttGAN used in this study is shown in The Unet-based generator  had subux from the previous encoding layer g and current decoding layer f were transformed by a convolution (1 × 1 × 1) respectively, where g(x) = Wgx and f(x) = Wfx. gW and fW were trainable parameters. The feature maps of decoding layer f(x) were then down-sampled to be consistent with the size of g(x). We performed an inner product of the two vectors g(x)and f(x) to obtain the feature dependencies between every two voxels:The attention modules, which were used for calculating the relationships of each voxel to all other pixels within a feature map, were incorporated together with the skip-connection between the encoding layers and decoding layers in the generator  20). Th. Th20). i,j further went through ReLU activation, convolution (1 × 1 × 1), and softmax function to normalize and reshape the feature maps to become ri,j. Finally, we multiplied ri,j with the feature maps x from the previous encoding layer g to obtain the attention coefficients Att:The α1L loss (LADV were used for training the generator g. The discriminator was trained by a cross-entropy loss DL (The L1 loss  and the  loss LD . The finLV1 (G) (where λ is set to be 100 to adjust the weight of VL1 (G) . The AttVL1 (G) , 19.All the intensity values of MP-SPECT projection images were normalized to a range of 0–1 for training. In addition, we further incorporated the binary patient defect information, i.e., with (1) or without defect (0) from patients’ own medical records, by embedding four 64 × 64 slices with the same binary values into the projection images .The AttGAN incorporating patient defect information (AttGAN-def), AttGAN, cGAN, and Unet were implemented using Tensorflow which ran on a NVIDIA GeForce RTX 2080Ti GPU. The Adam optimizer was applied to optimize this proposed model based on an initial learning rate of 0.0001 and trained to 400 epochs.We performed a 5-fold cross-validation on the clinical datasets to evaluate four DL approaches for various fast SPECT acquisitions. Specifically, for each fold of evaluation, FT and fast SPECT projection images of 35, 5, and 10 patients were selected for training, validation, and testing, respectively. This process was repeated 5 times and all fifty patient datasets were tested and averaged for the final results. The denoised projections were further reconstructed using the same OS-EM algorithm with 5 iterations and 4 subsets with attenuation and scatter correction. No post-reconstruction filter was applied on the reconstructed images generated from the denoised projections for further analysis.The hyper-parameters, e.g., number of layers and filters within each layer, were determined based on a training-validation procedure for AttGAN. Specifically, the number of layers varied as 2, 3, 4 and 5, while the number of filters within each layer varied as 8, 16, 24, 32 and 40. The hyper-parameters for cGAN and Unet were determined in our previous study , 19. TheThe voxel-based error of the denoised images was assessed by the normalized mean square error (NMSE), structural similarity index (SSIM), peak signal-to-noise ratio (PSNR), joint histogram, and linear regression measured on a 3D volume-of-interest  is the number of voxels in the VOI, μD and μFT are the mean values of the denoised and reference images, σD and σFT are the standard deviations of the denoised and reference images respectively, and σD,FT is the cross-covariance between the two images. The constants 1C and 2C are set to be 0.01 and 0.02, respectively  were drawn on the defect region and a uniform normal region on the polar plots based on visual assessment (1) divided by the mean value of the uniform ROI (ROI2). The absolute error of IR between FT images and different denoised images was computed.Two regions-of-interest , i.e., an index similar to the total perfusion deficit, was measured by the Wackers-Liu CQ™ (WLCQ) software  . The absThe Bland–Altman plots of PDS for different denoised methods and fast SPECT images are shown in 2= 0.8633), followed by the AttGAN (R2= 0.8433), cGAN (R2= 0.8341), and Unet (R2= 0.8123) on 1 s/prj fast SPECT denoised images.The voxel-based joint histogram and linear regression analysis results are shown in This study aims to assess the potential of attention-based DL network on fast MP-SPECT using clinical datasets. The attention module, inspired by non-local means, was proposed to enable the remote voxels to contribute to the local receptive filter during convolutional filtering . To the Our proposed networks were trained and tested for each dose-specific dataset, respectively, which were acquired on a CZT scanner. The denoising process is similar to an image-to-image translation task, and should be applicable to data acquired from other scanners or other denoised tasks, e.g., low dose SPECT imaging. The difference between low dose and fast SPECT is that the former will be more subject to patient motion with less radiation dose delivered to the patients. This is particularly important for the increasingly young patient population which has higher radiation risk than seniors . In our Shiri et al.  suggesteIn addition, some studies have shown that concatenating the gender, BMI, state (stress or rest), and scatter window images to the training dataset can improve the DL performance in the attenuation correction task . We firsRamon et al.  comparedLimitations of this study include that the clinical-related evaluation is only conducted by PDS. A more comprehensive clinical analysis and a ROC study of defect detectability are needed to validate the proposed methodology. Another limitation is that our DL networks were trained based on a relatively small patient cohort, i.e., thirty-five patients. Training on a large number of patients would benefit the model performance with less susceptibility of overfitting, though our network’s loss function was validated to be converged. Gong et al. proposed to pre-train the DL network with simulation datasets and then fine-tuned it with a limited clinical dataset . Their rIn this work, we investigated the performance of AttGAN in denoising fast MP-SPECT images using clinical datasets. The proposed AttGAN provided superior denoising performance as compared to the conventional cGAN and Unet. Patient defect information could be useful parameter for further improving the AttGAN-based denoising performance.The original contributions presented in this study are included in this article/supplementary material, further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by Institutional Review Board of Taipei Veterans General Hospital (IRB number 2022-11-002CC). The written informed consent was waived due to the retrospective nature of this study.JS, T-HW, B-HY, and GM were the primary authors of the manuscript. JS was mainly responsible for data processing and analysis. YD was mainly responsible for data analysis and manuscript revision. C-YL was mainly responsible for data collection. T-HW, B-HY, and Y-HL were responsible for clinical interpretation. GM and T-HW were responsible for data interpretation and study integration. All authors contributed to the article and approved the submitted version.


E-PS (trans) to Z-PS (cis), which transforms oblate ellipsoidal micelles intosmaller, spherical micelles with larger shell thickness. Doping PSwith CTAB resulted in mixed micelle formation at all stoichiometriesand conditions investigated; employing selectively deuterated PS,a monotonic variation in scattering length density and dimensionsof the micellar core and shell is observed for all contrasts. Theconcentration- and irradiance-dependence of the E to Z configurational transition was establishedin both neat and mixed micelles. A liposome dye release assay establishesthe enhanced efficacy of photosurfactants at membrane disruption,with E-PS exhibiting a 4-fold and Z-PS a 10-fold increase in fluorescence signal with respect to pureCTAB. Our findings pave the way for external triggering and modulationof the wide range of CTAB-based biomedical and material applications.The self-assembly of an arylazopyrazole-based photosurfactant(PS),based on cetyltrimethylammonium bromide (CTAB), and its mixed micelleformation with CTAB in aqueous solution was investigated by smallangle neutron and X-ray scattering (SANS/SAXS) and UV–vis absorptionspectroscopy. Upon UV light exposure, PS photoisomerizes from  These isomers display differentmolecular geometries, electronic absorption spectra, and dipole moments.9 Azobenzene motifs have been incorporated intosurfactant molecules11 and studied for their structure–function–assemblyrelationships.16 Light-induced switching of a photosurfactant’s conformation18 and properties19 has emerged as a powerful strategy for tuning the surfactant’samphiphilicity on demand.20Photoswitchable molecules transform betweentwo or more statesin response to light and have been employed in a variety of applications,ranging from energy storage21 have shown near quantitative E to Z switching,22 along-lived metastable state,23 and a largechange in the dipole moment.27 The latter feature has been recently exploited byKimizuka and co-workers to mediate the solubility of an arylazopyrazolein aqueous media.24 These properties facilitatehigh conversions between the two isomeric forms, while their long-half-livesallow their metastable and assembled structures to be studied anddeployed in applications. The arylazopyrazole’s improved photoswitchingproperties, paired with the greater structural change of the two isomericforms,23 render them as ideal candidates to incorporateinto photosurfactants.30 Despite showingpromise in controlling the assembly of supramolecular structures,36 relatively few studies have exploited the use of these azoheteroarenes.Specifically, an investigation into the key changes in aggregationbehavior occurring between the isomeric states of arylazopyrazoleshas not yet been conducted. Moreover, demonstrating the ability tomodulate the supramolecular assembly process using light, and characterizingthe emerging morphological changes to the system would provide anunderpinning framework for applications based on these light-responsivesurfactant systems.A subclassof azobenzene photoswitches, the arylazopyrazoles,Supporting Information (SI)). The structure of PS was designed to mimic the headgroup andchain length of the ubiquitous cationic surfactant CTAB ,we were able to monitor the precise shape and size of neat E-PS and Z-PS micelles, and mixed micelleswith CTAB, as a function of stoichiometry, concentration and illumination.Herein, we report the synthesis and solutionbehavior of a water-solubleamphiphilic photosurfactant (PS) that contains an arylazopyrazolemotif  attributed to the assembly of PS into micelles. The initial E-rich solution of PS was switched to a Z-rich photostationary state (PSS) by irradiating with 365 nm light. The metastable Z-state displayeda thermal half-life of 5.7 years at room temperature (25 °C),allowing us to infer that no appreciable thermal Z to E switching takes place over the course of ourmeasurements . The E to Z quantum yield of photoisomerizationfor PS using 365 nm irradiation was measured to be 0.61 .The synthesis of PS and deuteratedanalogues employed in our SANSstudies (PS-dH and PS-dT) is detailed in nm light1b, achieFigure S6 SI). We determined that the 365 nm PSS of a 1 mM solution of PScould be achieved within 15 min using a lamp of 20 mW/cm2, as no further changes could be observed in the UV–vis absorptionspectra. Using the same setup, volume, and concentration, subsequentsamples were irradiated for 20 min to ensure that the 365 nm PSS wasreached. In SAXS experiments employing higher PS concentrations (10–40mM), the irradiation dose was kept constant and the degree of isomerizationtracked by UV–vis absorption spectroscopy (Section 4 SI).Combining PS with varyingequivalents of CTAB did not yield anynoticeable difference in the energy of the electronic absorption bandsof the PS. Moreover, no detectable changes in the 365 nm PSS compositionwere observed for these samples  for CTAB is ∼1mM and is estimated to be ∼7 mM for PS from surface tensionand SANS experiments. Aqueous solutions in the concentration range2.5–40 mM were thus chosen for the scattering measurements. E-PS micellar solutionsas a function of concentration, exhibiting the expected profile forcharged micelles, analyzed in terms of form and structure factors.The effect of photoswitching of E-PS to Z-PS on the micellar size was studied by SAXS, as a function of concentration(10–40 mM) as shown in 37 The oblateellipsoids are characterized by an equatorial radius, Req (major) and a polar radius, Rp (minor): upon increasing the concentration (from 2.5 to 40mM), Req increases while Rp remains largely unchanged, indicating micellar growthalong the equatorial direction, and these values are in good agreementwith previous CTAB reports.39 The scattering profile of E-PS micelles also shows good agreement with an oblate ellipsoidalmodel for micelles  formed in solution by micelles 2b.E-PSmicelles yield alonger minor radius Rp and shorter majorradius Req, as compared to neat CTAB. E-PS micelles are overall smaller than those of CTAB, despitepossessing the same number of carbon atoms in the tail region , the overall smaller geometry of PS micelle is somewhatcounterintuitive if only the sterics of the two isomers is considered.Specifically, the bulkier arylazopyrazole moiety could result in alarger tail volume, characterized by a larger packing parameter andtherefore a larger micelle. We infer that the ability of the photochromicunit of PS to π-stack imposes a tighter packing of the hydrophobictails, leading to a geometrically constrained hydrophobic region.On the other hand, the aliphatic chain of CTAB exhibits significantlymore conformational freedom, thus resulting in a larger micellar size.The fitted dimensions of l region 2d. AlthoE and Z states were performed at concentrations above 10 mM, abovethe CMC of all surfactants. The profiles of E-PSmicelles were best fitted by oblate core–shell ellipsoidalmodel and the fitted parameters are in agreement with the values obtainedby SANS analysis. Combining SANS and SAXS, the composition of themicellar core and shell were estimated from the respective scatteringlength densities, and dimensions from independently fitting the sameform factor models , while SANS/SAXS provides greater discriminating power for theshape and size of the micellar ensembles.SAXS measurements for PS in both the r models 2d. Cryo-Z-PS are visibly differentand were described instead by a spherical core–shell micelle,indicating an oblate to sphere transition upon E to Z isomerization of PS  thebent “T-shape” conformation of the Z-PS structure prevents the π-stacking possible for E-PS25 and (ii) an increase inPS’s dipole moment in the tail portion of Z-PS.24The scattering profilesof on of PS 2d. The tZ-PS. This is consistent withthe molecular structure of E and Z-PS: the charged ammonium headgroup (hydrophilic) is separated fromthe arylazopyrazole (AAP) unit by a short butyl-chain; AAP possessesa dipole moment in the E-state, which increases uponthe configurational switch to the Z-state. To furtherprobe the difference in micellar polarity, Nile red-loaded micelleswere prepared and their fluorescence spectra were measured beforeand after exposure to light. The decrease in emission intensity observedfor Z-PS micelles relative to E-PSindicates dye release and increased polarity of the former . In addition to a change in themagnitude of the Z-PS dipole moment, its orientationalso changes. These changes in the dipole moment have the effect ofreducing the hydrophobicity of the tail region, and the hydrophilicheadgroup can be viewed as extending partially over the AAP unit.Further, the two isomeric forms ofPS exhibit differences in thepolar shell, inferred from the SAXS data, namely, an increase in theshell thickness for Z-PS, a reduction of the packing parameter, and correspondingreduction of equatorial radius . The findings are comparable with the self-assembly behaviorof gemini surfactants where headgroup repulsions result in largerseparations and give rise to a large effective headgroup area. Theresulting packing increases interfacial curvature and results in sphericalmicelles.41 The charge per micelle of CTAB is foundto be larger than that of either PS micelles, from analysis of thestructure factor, as expected given the larger micelle volume andaggregation number of CTAB. Upon illumination, the effective chargeof E-PS decreases considerably, as a result of thesurfactant structural change, which impacts the spatial arrangementof the micellar shell, as well as its shape .This leads to an increase in the effective headgroup area of the partial isomerization for samples illuminatedwith an insufficient UV dose for complete isomerization, at this (10–40mM) concentration range, viz. 10 min at 20 mW/cm2, correspondingto approximately 1/4 of that employed in the previous experiments.The resulting SANS profiles are shown in Req decreases while Rp increases, albeitmodestly , asexpected for partial isomerization of E-PS to Z-PS. As seen in Motivated by the practical application of thesesystems, we examinedthe effect of within CTAB mixed micelles, the mixed solutions were first prepared withthe E-PS state and subsequently illuminated priorto measurement. Our data show that PS:CTAB mixed micelles are formedat all ratios and with scattering length densities (SLDs) that varymonotonically with stoichiometry. E and Z states. Doping E-PS with CTAB resultedin micelles displaying intermediate structural dimensions betweenCTAB and E-PS, as expected for mixed micelles . In orderto examine the ability to photoswitch the PS micelles 4b. In thFigures S11 and S12 SI, are characteristic ofmicelle formation  and the corresponding SLDs are shownin In order to independently resolve the micellarcore–shellstructure, custom-synthesized selectively deuterated PS surfactantsolutions were measured by SANS. These contained either a deuteratedtail, PS-dT, or a deuterated charged headgroup, PS-dH. Various contrastsof mixed surfactant solutions were prepared, namely: PS:CTAB, PS-dT:CTABand PS-dH:CTAB at a 50:50 ratio, and 20 mM total concentration. TheSANS and dynamic light scattering (DLS) profiles, detailed in E-PS and Z-PS, referenced tothat of CTAB using a liposome permeabilization assay, detailed in Section 5 SI. This cell-free assay measures membranepermeabilization by encapsulating a fluorophore within liposomes andits subsequent release induced by the surfactant-mediated vesicledisruption. This assay has been successfully employed to investigatelipid bilayer leakage induced by intercations with nanoparticle42 and domains of viruses.43 Calcein (dye) enclosed liposomes were prepared  and their permeabilization was assessed inreal time by the increase in fluorescence intensity caused by dequenchingof the released dye into the surrounding buffer by the action of surfactants. E-PS, and Z-PS at both partial and fully converted state over time.Both E-PS and Z-PS at 1 mM concentrationshow a significant increase in dye release compared to pure CTAB andwithin barely 20 s reaction time, E-PS shows an approximately4-fold increase in fluorescence, while Z-PS showsa 10-fold increase, evidencing faster membrane disruption upon lightexposure. We interpret this observation in terms of the increasedpolarity and greater headgroup area of E-PS, andfurther of Z-PS, that improves respectively theirinsertion ability into the liposome bilayer, and their potential todisrupt lipid packing. The superior efficiency of Z-PS  core and thespacer form part of the effective hydrophilic segment of the molecule,and thus photoisomerism can be used to tune the physical propertiesof the surfactant. Liposome permeabilization  shows significant promise, demonstratingthat Z-PS acts significantly faster with greateryield (10×) than CTAB, exhibiting its superior efficacy as anantiseptic agent. Overall, these findings demonstrate the photoisomerizationpotential of the investigated PS in both neat and doped conditionsand highlight the importance of judiciously designing the PS’sstructure for a wide range of optically addressable applications.In summary, we have investigated the photoisomerismand self-assemblyof a model CTAB-based photosurfactant with an aryazopyrazole photoswitch,focusing on the impact of illumination on the neat and mixed micelleformation with CTAB. While CTAB and SI, alongsidewith accompanying DFT calculations, performed using Gaussian 16.The design and synthesisof the photosurfactant (PS) and head/tail deuterated analogues isdescribed in detail in the 1H NMR spectroscopy . Briefly, a single set of resonances was observed for the preirradiatedsamples and assigned to the E-isomer. Upon 365 nmirradiation, a new set of resonances appeared corresponding to the Z-isomer. Irradiation was performed until no further changein the integration of these two signals was observed, indicating thatthe PSS was obtained. NMR samples were then diluted, and a UV–visabsorption spectrum was recorded. Comparison of the UV–visabsorption spectrum of the pure E-isomer with thespectrum of the 365 nm PSS  served as a calibration for the UV–visabsorption experiments shown in this work. UV 365 nm irradiation wasachieved using a custom-built irradiation setup using 3 × 800mW Nichia NCSU276A LEDs.Photostationary states (PSS) were determinedusing –1, and wavevector Q range of 0.005–1 Å–1 achievedby two detectors at 2.4 and 4 m from the sample. Banjo quartz cellsof 1 mm path length were used for the measurements. Data were reducedand calibrated using MANTID, radially averaged, and analyzed withSasView (v5.0.4) using an ellipsoid form factor and Hayter mean sphericalapproximation (HMSA) structure factor. To achieve partial isomerization,samples were irradiated at 365 nm for 20 min and 20 mW cm–2.Measurementswere carried out on the time-of-flight SANS2D diffractometer at ISISpulsed neutron source , with an incident wavelengthrange of 2–14 ÅQ range 0.0031–0.34 Å–1. Sampleswere loaded into a 96-well PCR plate and stored at 30 °C beforeinjection into a quartz capillary, held at 30 °C, for measurement.All the samples were moved at 1 μL/s through the beam to avoidbeam damage. Samples were irradiated at 365 nm for over 6 h usingan LED lightbox operating at ∼6 mW cm–2,rotating the samples every hour.Measurements wereperformed at BioSAXS beamline B21, Diamond Light Source , at 12.4 keV and a detector distance of 4.014 m, yielding the Figure S13). A dye release assay wasset up with a total reaction volume of 200 μ L .Assay components were added directly to the wells of a 96-well solidblack plate, adding the surfactant last. The reaction volume was gentlymixed, and the plate was transferred to a Spectra Max M2 microplatereader with the excitation/emission set to 490/515 nm. Fluorescencedata were collected every 10 s for 10 min at room temperature, andnormalized.Phosphatidylglycerol(PG) and phosphatidylcholine (PC) lipids were dissolved in chloroformand mixed in a 1:1 molar ratio at 5 mg/mL concentration. The solventwas then evaporated to create a thin film and placed under vacuumfor at least 1 h. Phospholipid films were hydrated to a 100 mM aqueoussolution of calcein containing NaOH (added to facilitate completedissolution). The flask was swirled gently for 30 s every 5 min for1 h to ensure encapsulation of calcein into the core of liposomes.Liposomes were then extruded 21 times through a polycarbonate membrane(200 nm pore size). During preparation, liposomes were maintainedabove the phase transition temperature of lipids (25 °C is acceptablefor 1:1 (mol/mol) PG:PC). Calcein enclosed liposomes were separatedfrom free dye by Sephadex G-50 beads column using 1× PBS buffer.Dynamic light scattering and fluorescence spectroscopy confirmed theformation and size uniformity of liposomes (average diameter of 100nm,


Access to screen-based media has been revolutionized during the past two decades. How this has affected sedentary time (ST) accumulation in children is poorly understood.n = 1722), 2011 (n = 1587) and 2018 (n = 1859). Secular changes between surveys were analysed using random-effects linear regression models adjusted for survey-specific factors. Data on ST were collected using hip-worn ActiGraphs and ST was defined using a threshold equivalent to <100 counts/min. Sedentary bouts were grouped by duration: <1, 1–5, 5–15, 15–30 and ≥30 min.This study, based on the Physical Activity among Norwegian Children Study (PANCS), uses accelerometer data from population-based samples of 9- and 15‐year-olds, collected in 2005 (P <0.001), by 21 min/day in 15-year-old boys  and by 22 min/day in 15-year-old girls , but not in 9-year-old girls at 6 min/day . All age-sex groups accumulated less ST in bouts lasting <5 min and more ST in longer bouts, particularly in 5–15-min bouts. Adolescent girls also increased ST accumulation in 15–30-min and ≥30-min bouts. Changes were largely mirrored before, during and after school on weekdays and during weekend days.Between 2005 and 2018, ST increased by 29 min/day in 9-year-old boys (95% CI: 19, 39; Coinciding with the introduction of smartphones, tablets and near-universal internet access, total daily ST and ST accumulated in prolonged sedentary bouts increased between 2005 and 2018 in children and adolescents. Key MessagesSedentary time is a risk factor for morbidity and early mortality, with prolonged bouts of sedentary time suggested as particularly detrimental.Technological advancements have revolutionized access to screen-based media during the past two decades, but how this has affected the total volume and accumulation pattern of sedentary time in children and adolescents is poorly understood.We used accelerometer data from population-based samples of 9- and 15‐year-olds collected in 2005, 2011 and 2018, to examine secular changes in sedentary time from before to during and after the introduction of smartphones, tablets and near-universal internet access.We found that sedentary time increased by 20-30 min/day in 9-year-old boys and in 15-year-olds from 2005 to 2018, and that all age-sex groups spent less time in sedentary bouts lasting <5 min and more time in longer bouts of uninterrupted sedentary time.Our data suggest that contemporary children and adolescents are spending a larger part of their day in uninterrupted sedentary pursuits than before the introduction of smartphones, tablets and near-universal internet access.A high level of sedentary time (ST) is a risk factor for morbidity and early mortality.The advent of available and affordable technology and digital communication during the past 20 years is considered to have fundamentally changed the way children and adolescents engage in screen-based behaviours.The Physical Activity among Norwegian Children Study (PANCS) is a serial, cross-sectional survey of accelerometer-assessed physical activity and ST, designed to monitor secular changes between nationally representative samples of children and adolescents, and serves as the national physical activity and ST surveillance system in Norway. Because the first survey was initiated in 2005, we are in a unique position to perform a detailed comparison of ST before (2005), during (2011) and after (2018) the introduction of smartphones, tablets and near-universal internet access. We therefore aim to describe secular changes in total and patterned ST in 9- and 15-year-old children and adolescents. As a secondary aim, we explore if changes are similar on weekdays and weekend days and during time-segments on schooldays .Three waves of the Physical Activity among Norwegian Children Study have been conducted by the Norwegian School of Sport Sciences in collaboration with the Norwegian Directorate for Health and the Norwegian Institute of Public Health, in 2005 (PANCS1), 2011 (PANCS2) and 2018 (PANCS3). All study waves were carried out in accordance with the Declaration of Helsinki. The Regional Committee for Medical Research Ethics (RCMRE) approved PANCS1, whereas PANCS2 and PANCS3 were considered outside the Health Research Act's scope by the RCMRE and therefore not considered subject to approval. The Norwegian Centre for Research Datan = 1119), or from a random sample of schools that had previously participated in PANCS1 (n = 640). In 2018 (PANCS3), we invited the same schools that Statistics Norway had selected for PANCS1 (10th graders) and PANCS2 (4th graders). If a school declined to participate, we invited another school from the same, or a corresponding, geographical and sociodemographic area. When schools agreed to participate, we invited all 4th and/or 10th grade pupils to participate. The present study compares cross-sectional data from 9- and 15-year-olds participating in PANCS1, 2 or 3.In 2005 (PANCS1), Statistics Norway selected nationally representative samples of 9- and 15-year-olds (4th and 10th graders) using cluster sampling with schools as the primary unit, taking geography and population density into account. In 2011 (PANCS2), a mixed design was used. Statistics Norway selected a new, nationally representative sample of 9-year-olds using the same method as in PANCS1. Further, 15-year-olds were either invited individually, based on previous participation in PANCS1 at age 9 years . PANCS1 used model CSA7164, PANCS2 used models GT1M and GT3X+, and models GT3X+ and GT3X+BT were used in PANCS3. Participants were instructed to wear the monitor during all waking hours for a full week in PANCS2 and 3, and for 4 consecutive days (including 2 weekend days) in PANCS1, due to limited monitor storage capacity of the CSA7164. All monitors were initialized to start recording at 06:00 the day after the participants received them.IJE online, page 2).The piezoelectric accelerometer within ActiGraph model CSA7164 records less ST than the Micro-electromechanical system-based accelerometers within the newer generations of ActiGraphs under default settings.After excluding all data recorded between 00:00 to 06:00 and non-wear (defined as consecutive spells of zero counts lasting ≥60 min),We classified all 10-s epochs with <17 activity counts as ST, which is equivalent to the widely used ≤100 CPM cut-point.We considered age, seasonality (minutes of daylight on accelerometery start date), mean accelerometer wear time (min/day or min/segment) and weekday:weekend ratio  as relevant covariates.,,Secular trends in total ST and bouts of ST across the 2005, 2011 and 2018 surveys were analysed using random-effects linear regression, with school as the random effect and 95% confidence intervals adjusted for the school-level (cluster) sampling. All secular trend analyses were adjusted for age, minutes of daylight at the time of ST assessment and segment-specific accelerometer wear time as fixed effects, because between-survey differences in these variables may confound estimates of ST.Among 5261 and 5421 invited 9- and 15-year-olds overall, 4072 (77%) and 3331 (61%) agreed to participate, respectively . After eP <0.001) and 2011 at 17 min/day , with increases observed on both weekdays and weekends. The mean difference in ST observed between 2005 and 2018 in 9-year-old girls was much smaller (6 min/day), and the 95% confidence interval included unity .Nine-year-old boys and girls spent an average of about 7 h 45 min sedentary per day in 2018 . In boysP = 0.002) to an average of 9 h 36 min/day in boys, and in girls by 22 min/day  to an average of 9 h 58 min/day (P <0.001) than on weekend days at 10 min/day . In girls, the increase in ST was similar on weekdays and weekend days between 2005 and 2011, with no further increase observed between 2011 and 2018 overall at -2 min/day .In 15-year-olds, ST increased overall among boys and girls between 2005 and 2018, by 21 min/day  suggests that secular increases have been larger at the lower end of the distribution in 9-year-old boys and 15-year-old girls. Hence, in these groups, fewer children and adolescents are now at the lower end of the ST distribution. We did not have access to comparable information on parental education or income across surveys, which prohibits us from examining secular changes across socioeconomic gradients.Device-based measurements of ST in large epidemiological studies have become feasible within the past two decades. Yet, such data are only available in a few population-based surveys and even fewer have data covering multiple cycles, a prerequisite of monitoring secular trends.Self-reported ST from the National Health and Nutrition Examination Survey (NHANES), operationalized as total sitting time, suggests an increase in US adolescents’ ST between 2007 and 2016 of 1.1 h/day .It is difficult to establish if an average increase in ST of 20 to 30 min/day in children and adolescents is a cause for concern. Currently no quantitative target is given for total ST, and the health impacts of more ST is likely determined by the behaviour that is being replaced, with more evidence to support adverse consequences of less time spent in moderate-to-vigorous physical activity.,,,Currently, the combined evidence from literature reviews prepared for the 2020 WHO Physical Activity Guideline Development Group suggested that children and adolescents should limit ST in general, and particularly ST accumulated from recreational screen-time.It is insufficiently understood whether prolonged sedentary bouts are a particular health risk in children and adolescents, above and beyond the total volume of ST.We highlight the following limitations. The PANCS surveys do not have sampling weights available to account for a potential systematic bias in non-participation across socioeconomic or geographical characteristics. Similarly, we do not have access to data allowing a formal comparison of our samples with characteristics of the general population. Registry-based parental education was available in PANCS2, suggesting no socioeconomic bias.Hence, we believe our main message of increases in ST over the past 12 years is robust to potential non-identical participation mechanisms across surveys. Also, the participation rate among 9-year-olds can be considered high across surveys. Hip-worn accelerometers are unable to discern sitting, lying or reclining from standing which may lead to some misclassification. This would bias estimates of secular changes in ST if standing behaviours have changed.In conclusion, from 2005 to 2018, ST has increased by almost 30 min/day in 9-year-old boys, has been stable in 9-year-old girls, and has increased by about 20 min/day in 15-year-old boys and girls from Norway. Children and adolescents accumulate more of their ST in bouts lasting longer than 5 min than they did before the introduction of smartphones and ubiquitous internet access.The Regional Committee for Medical Research Ethics (RCMRE) approved PANCS1 (REK Sør: S-04305), whereas PANCS2 (REK Sør-Øst: 2010/3127) and PANCS3 (REK Sør-Øst: 2017/1426) were considered outside the Health Research Act's scope by the RCMRE and therefore not considered subject to approval. The Norwegian Centre for Research Data approved the processing of personal data in all three study waves .The approval from the Ethics Committee does not include permission to make data materials publicly available. Analytical code (Stata do-files) will be made available upon request.IJE online.K.E.D. (guarantor) and J.T. had the idea for and designed the study, pooled and linked the data, analysed the data with help from J.S.S., B.H.H. and EK and take responsibility for its integrity and the data analysis. K.E.D. and J.T. wrote the first draft of the report. All other authors assisted in developing the statistical models, reviewed results, provided guidance on methods and critically reviewed the manuscript. All authors read and approved the final version of the manuscript.This work was supported by the Norwegian Research Council (grant number 249932/F20) and by the Department of Sports Medicine, the Norwegian School of Sport Sciences.dyac063_Supplementary_DataClick here for additional data file.


Chronic stress impairs working memory (WM), but few studies have explored the protective factors of the impairment. We aimed to investigate the effect of self-awareness on WM processing in people under chronic stress. Participants under chronic stress completed an n-back task after a self-awareness priming paradigm during which electroencephalograms were recorded. The behavioral results showed that participants whose self-awareness was primed reacted faster and more accurately than the controls. Event-related potentials (ERPs) revealed the following (1) P2 was more positive in the self-awareness group than in the controls, indicating that self-awareness enhanced allocation of attention resources at the encoding stage. (2) N2 was attenuated in the self-awareness group compared with the controls, indicating that smaller attention control efforts were required to complete WM tasks adequately after self-awareness priming; and (3) enhanced late positive potential (LPP) was evoked in the self-awareness group compared with the controls, suggesting self-awareness enabled participants to focus attention resources on the information at the maintenance stage. Critically, mediational analyses showed that LPP mediated the relationship between self-awareness and WM response times. This result suggests that the fact that participants whose self-awareness was primed were able to achieve better behavioral performances may be attributed to their mobilization of sustained attention resources at the maintenance stage. In summary, self-awareness exerted a protective effect on WM in those under chronic stress, which may be due to the enhancements in the allocation and mobilization of attention. These results could be used to develop more specific coping strategies for people under chronic stress. Working memory (WM) is a fundamental process underlying higher cognitive functions, such as learning, language comprehension and problem solving. Thus, WM has an important significance to an individual’s adaptation and survival . AbundanSelf-awareness refers to a state in which individuals pay attention to themselves and experience heightened awareness of their own internal feelings and beliefs . The curAlthough some people are more self-aware than others in trait  , self-awEvent-related potentials (ERPs) have been widely used to assess alterations in the dynamic time course of neural activity during WM processing . WoodmanIn summary, the purpose of the current study was to explore the influence of priming self-awareness on WM in people under chronic stress. Participants were divided into two groups (self-awareness condition and control condition), and completed the implicit self-awareness priming task  or control task, respectively. Subsequently, all participants were asked to complete an n-back task, during which time behavior and electroencephalogram (EEG) were measured . Based ot = −0.51, p > 0.05). There was no significant difference between the two groups with respect to PSS . Additionally, to exclude the possible intellectual differences between the two groups, we assessed participants’ IQ using the Raven’s Standard Progressive Matrices . The study and recruitment of participants were approved by the Ethics Committee of the local university and performed in strict accordance with the approved guidelines.The present study specifically sought to explore the impact of self-awareness on WM in people under chronic stress. The relationship between self-awareness and WM among people with a low stress level will be reported elsewhere. Participants were recruited from a local university through online and public postings. All postings specified the inclusion criteria of the study: (1) graduates who were preparing for the CNPEE  from May 2021 to December 2021 because many previous studies have supported that examination preparation is a long-term stressor ; (2) phyPerceived stress was assessed by Cohens PSS (10-item version) . The scaSLSI  is a selAfter arriving at the laboratory, participants were informed of the experimental procedure and completed the demographic information collection. After application of the electrodes, participants were seated in a dimly lit, sound-attenuated, electrically shielded chamber with a CRT monitor set approximately 60 cm away from participants’ eyes. Participants completed the SST and n-back task while behavioral and EEG data were recorded.Participants were first asked to finish the SST to increase self-awareness . In the After the SST task were finished, a spatial n-back task was immediately used to test WM . Task diThe reaction times (RT) and accuracy (ACC) data for the spatial n-back task were included only when the WM probe was correctly recognized. Removal of these trials resulted in the elimination of 6% of all trials. A repeated-measure analysis of variance (ANOVA) was performed with two (group: self-awareness vs. control) between-subject factors, and three  within-subject factors.Brain electrophysiological activity was continuously recorded from a 64-Channel EEG recording system  with references on the middle at FCz. The inter-electrode impedance was always controlled below 10 kΩ. EEG and EOG were amplified using a 0.05–100 Hz band pass filter and continuously sampled at 500 Hz for offline analysis.t = −1.41, p = 0.29; 1-back: t = −0.06, p = 0.96; 2-back: t = 0.48, p = 0.68).Raw EEG data was processed offline using Brain Vision Analyzer version 2.1 . For the data analysis, (I) the former reference FCz was reinstated as an additional data channel ; (II) thBased on previous studies of n-back tasks, P2, N2, and LPP were measured . The folIn order to explore whether self-awareness has a protective effect on WM in people under chronic stress, and the neural mechanism involved, correlational analyses involving associations between the PSS, behavioral performance measures , and P2/N2/LPP were conducted. Moreover, the mediation model was tested to examine whether neural processing (P2/N2/LPP) mediated the expected relationships between self-awareness and behavior. In the model, the groups (SA/CC) were entered as the independent variable, P2, N2 and LPP as the mediators respectively, and the dependent variable was the behavioral performance  of the 0-, 1-and 2-back tasks, respectively. The analyses were performed using the SPSS macro from F = 102.84, p < 0.01, η2 = 0.69]. Post hoc analysis showed ACC of 0-back  was significantly higher than ACC of 1-back  and 2-back . ACC of 1-back was significantly higher than 2-back, p < 0.01. There was a main effect of group, , showing higher accuracy in SA compared with CC. The interaction of load and group was also significant . Simple effect analysis showed at 2-back load, the ACC of self-awareness group was significantly higher than those of control group , while there were no significant differences at 0-back  and 1-back   [.50] see -ACC.F = 16.92, p < 0.01, η2 = 0.27]. Post hoc analysis revealed that RT of 2-back  was significantly longer than RT of 0-back  and 1-back . There was no difference between 0-back and 1-back, p = 0.33. The main effect of group was significant , showing shorter response times in SA participants compared with controls. The load × group interaction effect was marginally significant . Simple effect analysis showed that in comparison with 0-back task , the response time of self-awareness group was significantly faster than control group at 1-back  and 2-back  loads  = 14.98, p < 0.01, η2 = 0.25]. Post hoc analysis showed that 2-back  evoked smaller P2 than 0-back  and 1-back . There was no difference between 0-back and 1-back, p = 0.66. The main effect of group was significant , showing increased P2 in SA participants compared with controls. The group × load interaction was not significant   = 25.64, p < 0.01, η2 = 0.36]. Post hoc analysis showed that 0-back  evoked larger N2 than 1-back  and 2-back . The amplitude of 1-back was significantly larger than 2-back, p < 0. 01. The main effect of group was significant  = 4.11, p = 0.04, η2 = 0.08), showing a smaller N2 amplitude in SA than it in CC. The group × load interaction was not significant   = 4.63, p = 0.01, η2 = 0.09] was significant. Post hoc analysis revealed that 1-back  evoked more positive LPP than 2-back , p < 0.01. There was no difference between 0-back  and 1-back, p = 0.10, as well as 0-back and 2-back, p = 0.31. For the main effect of group , we observed significantly increased LPP amplitudes in SA individuals than in controls. The group × load interaction was also not significant  . However, there was no significance in SA . Follow-up analysis revealed the same positive relationship found in N2. The N2 under CC was significantly positively correlated with PSS , while not significantly associated with PSS in SA . See We found a significant positive relationship between self-perceived stress and P2 in CC, and higher PSS scores were associated with larger P2 . 95% confidence intervals (CI) were . Meanwhile, LPP also significantly predicted the RT at 1-back . 95% CI was . The indirect effect of awareness group on RT was significant, 95% CI was , while the zero was included in 95% CI of direct effect . For 2-back load, awareness group (Self-Awareness and Control) significantly predicted LPP . 95% confidence intervals (CI) were . Furthermore, LPP also significantly predicted the RT  loads. 95% CI was . The indirect effect of awareness group on RT was also significant, 95% CI was . The zero was included in 95% CI of direct effect , which was not significant. See The mediation results showed a complete mediating effect of LPP on the association between awareness group and RT at 1-back and 2-back loads, respectively. Specifically, for 1-back load, awareness group (Self-Awareness and Control) significantly predicted LPP , indicating that individuals who have higher stress perception will evoke larger P2/N2, the self-perceived stress plays a critical role in the modulation of P2/N2 components for individuals who are under chronic stress. Those results are supported by a prior n-back task study . HoweverThe mediation analyses revealed that LPP mediated the relationships between the self-awareness-group and the RT of both 1-and 2-back tasks. LPP has been linked to memory maintenance, reinterpretation of stimulus meaning, as well as attentional biases as measured by RT . EvidencThe key contribution of this work is that it is the first attempt to combine self-awareness, WM, and chronic stress and provides a new idea for the protection of WM capacity in individuals under chronic stress. We verified that self-awareness indeed endows individuals with higher WM performances. Furthermore, with the advantage of high temporal resolution in ERPs, we explored the protective mechanism of self-awareness on WM capacity at the neural dynamics level. Exploring the protective role of self-awareness in WM processes in people under chronic stress is helpful to have a deeper understanding of individuals under chronic stress, so as to develop specific training and treatment programs.There are some limitations to the current study. First, our data were taken only from the results of one single intervention; however, the effects of chronic stress are long-term. Our modulation pathway would follow from longitudinal data and improve the level of trait self-awareness through a longer-term training. Second, we used scrambled sentences for priming self-awareness in the current study. In future research, a variety of paradigms and techniques for priming self-awareness should be developed. Other techniques, such as simultaneous EEG-fMRI and MEG, can be used, which would enable us to more directly examine the interplay of the PFC, self-awareness and WM tasks in people under chronic stress. Lastly, although the present work provides a first look at the protective effect of self-awareness for WM capacity, future research with larger and more representative participant samples are needed to establish a better understanding of these effects.We found that self-awareness protects WM from the adverse effects of chronic stress, which was accomplished by enhancing allocation and mobilization of attention. We found significantly better performance in self-awareness group participants, measured by accuracy and reaction times. The increase in P2, decrease in N2, and increase in LPP were consistent with our hypothesis. These findings suggest that the protective effect of self-awareness may occur at the encoding and maintenance stages in the WM process. Mediational analyses showed that the LPP mediated the relationship between awareness condition and WM performance . This result potentially suggests that the neural mechanism of the protective effect of self-awareness on WM in people under chronic stress was exerted by improving maintenance processing.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics Committee of Qilu Normal University. The patients/participants provided their written informed consent to participate in this study.ZW and YL had substantial contributions to the conception and design of the work. SZ and SH had substantial contributions to the acquisition, analysis, and interpretation of data for the work. DJ, SH, and YL had revising it critically for important intellectual content. WX participated in the whole process. All authors contributed to the article and approved the submitted version.This work was supported by NSFC (31920103009), the Major Project of National Social Science Foundation (20&ZD153), and Shenzhen-Hong Kong Institute of Brain Science -Shenzhen Fundamental Research Institutions (2022SHIBS0003).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The identification of biomarkers for neurodegenerative disorders such as Huntington’s disease (HD) is crucial for monitoring disease progression and therapeutic trial outcomes, especially in the pre-manifest disease stage (pre-HD). In a previous study, we observed that leukocyte telomere length (LTL) was strongly correlated with the estimated time to clinical onset in pre-HD subjects. To validate this hypothesis, we designed a follow-up study in which we analyzed LTL in 45 pre-HD stage subjects at baseline (T0) and then again after clinical onset at follow-up (T1); the follow-up interval was about 3 years, and the CAG range was 39–51 repeats; 90 peripheral blood mononuclear cell samples (PBMCs) were obtained from the Enroll-HD biorepository. In pre-HD subjects at T0, LTL was significantly reduced by 22% compared to the controls and by 14% from T0 at T1. No relationship was observed between the LTL and CAG numbers in subjects carrying different CAG repeats at T0 and at T1, suggesting that LTL reduction occurs independently of CAG number in pre-HD subjects. ROC curve analysis was used to test the validity of LTL as a potential biomarker of HD progression and showed that LTL measurement is extremely accurate in discriminating pre-HD subjects from the controls and even pre-HD from manifest HD, thus yielding a robust prognostic value in pre-HD subjects. Huntington’s disease (HD) is an autosomal dominant, progressive neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the first exon of the HTT gene encoding huntingtin (HTT), a ubiquitously expressed protein involved in transcriptional regulation ,2. The mThe classic signs of HD are chorea, cognitive decline, and behavioral and psychiatric disturbances . HD is fIn individuals carrying the mutation, symptoms gradually emerge during a pre-manifest phase (pre-HD), but the timing of the transition from pre-manifest to manifest status is difficult to establish. Predicting disease progression in the pre-HD stage would be a relevant achievement as it could facilitate better planning of possible therapeutical interventions. Statistical models have been devised to estimate the age at disease onset from the length of an individual’s CAG repeat, but their accuracy is limited because the number of CAGs accounts for only a part of the variation in the onset of the disease ,9. OtherHuman telomeres consist of tandem repeated TTAGGG nucleotide sequences located at the ends of chromosomes where they act as natural protective caps against DNA damage. During physiological DNA replication, telomeres progressively shorten with each cell division, due to the inability of the DNA polymerase to replicate the 3′ end of the DNA strand. Telomerase, a cellular ribonucleoprotein enzyme complex, counteracts telomere shortening. Though usually present in the early stages of embryonic development, its activity is almost absent in adult tissues, including skin, kidney, liver, blood vessels, and peripheral leukocytes ,13. As aPeripheral blood mononuclear cells (PBMCs) are ideal for telomere research; they are easy to obtain from blood and are readily available. Furthermore, because they circulate throughout the body, immune cells are exposed to both internal (from cell) and external (from diet and exposures) factors affecting telomere maintenance. They are also involved in the interaction between psychological processes and the nervous and immune systems . ShortenLTL in HD has been investigated in several studies and found to be shorter in HD patients than in controls ,29,30,31p < 0.0001), as were the differences between the HD patients at baseline and at follow-up, i.e., between the pre-manifest and manifest HD stages (p < 0.0001). A reduction in LTL of 0.13 ± 0.05 T/S, or about 0.04 T/S per year, between T0 and T1 was observed. LTL expressed as a T/S ratio was measured in 45 controls  and in patients at T0 (pre-HD stage)  and at T1 (manifest HD)  . The difp = 0.003; 95% confidence interval [CI] −0.00409 ≤ β ≤ −0.00111). No relationship was observed between LTL and age at baseline (pre-manifest HD) (p = 0.20), while there was a positive relationship between LTL and age at follow-up (manifest HD) where, however, the 95% CI of the regression coefficient was quite large . A statistically significant relationship was observed between the follow-up interval and the LTL reduction , indicating that the longer the follow-up, the greater the reduction in LTL. As LTL usually decreases with age in normal subjects, we analyzed the LTL/age relationship in the three samples . As expep < 0.05). We also wanted to determine the differences in LTL variation between HD patients carrying a different number of repeats. No difference in the median LTL of pre-manifest HD patients carrying different CAG repeat numbers was noted at about 2.5 years before disease clinical onset, even if the CAG repeat number was associated with a different age Tab 2, A, due top = 0.016)  .We then applied ROC curve analysis to test the accuracy of LTL as a biomarker. ROC analysis showed that an LTL cut-point of 0.895 (the Youden index criterion) had a sensitivity of 1.000 and a specificity of 0.956 (AUC 0.997) for distinguishing the pre-HD patients from the controls A. Comparp < 0.0001). At follow-up (T1), approximately 1 year after the clinical onset of HD, LTL values were further reduced by 14% from the baseline and 34% from the control LTL values. LTL in HD mutation carriers was examined in several studies [Our previous cross-sectional study suggested LTL as a good biomarker of HD conversion from the pre-manifest stage to overt disease . To conf studies ,29,30,31 studies  reported studies , spinoce studies . There i studies ,33. ). Similarly, no relationship was observed between LTL and age in pre-HD stage patients at T0, while a slight positive trend was found in manifest-HD-stage patients at follow-up  indicated that telomere length depended on age and CAG number . LTLs amThe LTL at follow-up, on average at about 1 year after the disease onset, was 66% of that of the controls, with a reduction of 0.13 T/S compared to the pre-HD stage, i.e., a telomere loss rate of about 0.04 T/S per year of follow-up. This rate is higher than the estimated rate of 0.01 T/S/years reported in healthy adults . PreviouWe noted no correlation between LTLs in the pre-HD or the HD stages and the clinical measures (TMS and TFC) ; howeverThe role of LTL as a potential biomarker of HD progression, as validated with ROC analysis, showed a high accuracy of LTL in discriminating the pre-HD patients from the controls (AUC = 0.997), and even in discriminating pre-HD from manifest HD (AUC = 0.979). In the latter case, setting the cut-point of LTL at 0.700 T/S enables manifest HD to be correctly predicted at the initial stages with a sensitivity of 0.911 and a specificity of 0.978. We also compared the diagnostic performance of LTL with mHTT and neurofilaments (NfLs) from cerebral spinal fluid (CSF) and NfLs from peripheral blood, as described in Byrne et al., 2018 . LTL seehttps://www.enroll-hd.org/)  Enroll-HD Biosamples Use Agreement Version RevNo002 (101216)). Two samples for each patient were collected during two visits about 3 years apart, for a total of 90 PBMC samples. The first sample at baseline (T0) was collected in an HD subject clinically classified at the pre-manifest stage in the Enroll database (pre-HD), and then the second sample at follow-up (T1) when the same patient was classified as manifest HD. The actual mean follow-up interval between T0 and T1 collection was 3.4 ± 0.7 years. The repeat range of mutated alleles varied from 39 to 51; there were about six patients per CAG repeat number , as the aim was to determine the differences in LTL variation between HD patients carrying a different number of repeats. Scores assessing motor function  and functional capacity  were obtained from the Enroll database. Age at clinical onset was defined as the age at which progression from the pre-manifest to the manifest stage was clinically established, as reported in the Enroll database. The control samples, matched for age at baseline and sex, were healthy blood donors recruited at the Department of Experimental Medicine and Surgery at the University of Tor Vergata, Rome, Italy. The use of the control subjects sample was approved by the CNR Research Ethics and Integrity Committee .The PBMCs of 45 HD patients, non-renewable material, were obtained from the ENROLL  according to the manufacturer’s instructions. Leukocyte telomere length was measured by monoplex real-time PCR quantitative analysis (monoplex qPCR) on a 7300 real-time PCR instrument . This method allows the number of copies of telomeric repeats (T) to be determined, as compared to a single-copy gene (S) used as a quantitative control (T/S ratio)  . TelomerData are presented as mean ± SD unless otherwise indicated. Data from paired groups  were compared using the two-sided Wilcoxon signed-rank test, while the unpaired data were compared using the Mann–Whitney test. Correlation was assessed using linear regression and Spearman’s correlation index. Multiple independent groups were compared using the Kruskal–Wallis test and post-hoc analysis. Cut-off points for the receiver operating characteristic (ROC) curves were determined using the Youden index criterion. Statistical analysis was performed using R-Bioconductor  and Statistix 8.0 . Our previous work  suggesteThere are no effective disease-modifying treatments for HD to date, but therapeutical trials investigating therapies to modify the course of the disease have shown promising results. There is, therefore, the need to identify among pre-manifest patients a temporal window more or less close to clinical onset in which to initiate disease-modifying therapies. Overall, our data show that LTL measurement has a robust prognostic value in pre-manifest HD, although it may be of limited relevance for tracking disease progression after clinical onset. Compared to other validated HD biomarkers, LTL measurement, obtained by a non-invasive procedure, may provide an ideal biomarker of HD disease progression .